Conjugation has been widely used to optimize the properties of biologically active proteins, such as protein/peptide therapies, antibody-drug conjugates, vaccines, tissue selective targeting vehicles, molecular diagnostics, and protein nucleic acid conjugates.
Traditional conjugation method utilizes lysine based covalent ligation, which is difficult to achieve homogenicity due to the abundance of lysines on the surface. The other typical method is based on cystein conjugation. However, cystein is a rare amino acid and usually present in disulfide form, and free cystein can potentially lead to disulfide scrambling.
Tyrosines represent better alternative conjugation sites. They are rarer than lysines, and not all of them are surface exposed. A few methods have been reported recently for tyrosine ligation, such as allylic alkylation, diazo-coupling and Mannich-type coupling. See, for example, J Am Chem Soc, 128, 1080-1081 (2006); J Am Chem Soc, 127, 3718-3723 (2005); and Bioconjugate Chem, 19, 153-157 (2008)).
A tyrosine ligation method was recently described by Ban, H., et al. in J Am Chem Soc, 132, 1523-1525 (2010). However, the predominant product observed using the published protocol (a Phosphate Buffered Saline (PBS) solution) is most likely urea formation at lysines. The LC-MS in the publication is inconsistent with the proposed tyrosine ligation product.